Fig 1.
Simplified diagram outlining current understanding of plant phylogeny, including species discussed in this publication.
Hornworts, Liverworts and Mosses make up the bryophytes, non-vascular land plants which reproduce via spores. Lycophytes are vascular spore producers and have structures called microphylls rather than true leaves. The dicot Amborella trichopoda is considered a representative basal angiosperm, with a primitive form of xylem tissue compared to other flowering plants.
Fig 2.
Alignment of representative PPD peptide sequences.
PEAPOD proteins contain three regions that are highly conserved; these are indicated by the yellow background (PPD domain), blue background (TIFY domain) and green background (truncated Jas domain). Sequences include: A. thaliana (accession NP_567442.2), S. moellendorffii (accession XP_002964672), A. trichopoda (accession XP_006838952.1), P. abies (accession MA_99597G0010, congenie.org), T. repens (accession MZ736871) and M. acuminata (accession M0S6C8_MUSAM, Uniprotkb).
Table 1.
Sequence identity across full sequences and distinct domains of PPD orthologues.
Fig 3.
Schematic diagram illustrating syntenic comparison of PPD and flanking genes in dicotyledons and monocotyledons.
Arrows indicate chromosome strand; where appropriate, the gene order has been reversed for easy graphical view. PPD1 and PPD2 are both shaded purple to signify they are very similar and in cases where only one orthologous protein was found in another organism it may represent either of the two genes. Similarly for ATARD1 and ATARD2. Only the closest syntenic clusters relative to the PPD gene are shown for G. max; S. lycopersicum, V. vinifera and M. acuminata.
Fig 4.
The Arabidopsis thaliana Δppd mutant was complemented with PPD from a broad range of multicellular land plants.
In Arabidopsis the Δppd mutant has a distinct propeller shaped rosette with transversely domed leaves (A) and dimpled, short paddle shaped siliques (B); these contrast to the WT rosette of straight, transversely flat leaves (C) and smooth, elongated torpedo shaped siliques (D).Over-expression of PPD from A. thaliana (E & F), and orthologues from S. moellendorffii (G & H), P. abies (I & J), A. trichopoda (K & L), M. acuminata (M & N), and T. repens (O & P) all complemented the Δppd mutant. Plants shown are homozygous lines for each construct. Scale bars = 10mm.
Fig 5.
Immunoblot analysis of recombinant C-terminally V5 tagged PPDs expressed in the leaves of the Δppd mutant A. thaliana.
Equal quantities of crude protein extract (bottom panel) from 17–19 day old rosette leaves of wild type A. thaliana (Ler) (A); Δppd mutant (B); Δppd mutant transformed to over express the PPD orthologue from: S. moellendorffii (C); P. abies (D); A. trichopoda (E); A. thaliana (F); M. acuminata (G) and T. repens (H) were subjected to PAGE-immunoblot. The membrane was probed using the anti V5 antibody (Life Technologies). No V5 signal was detected in the WT and Δppd mutant lanes while signals of the appropriate sizes were detected in transformed Δppd mutants over-expressing a PPD orthologue (top panel).
Table 2.
Ratios of complemented Arabidopsis Δppd plants with constructs containing optimised PPD sequences from various species.
Fig 6.
Alignment of Arabidopsis PPD1 with putative Kobresia and Joinvillea PPD proteins.
The alignment shows the conserved PPD (yellow), TIFY (blue) and Jas* (green) domains.
Fig 7.
Comparison of the protein domains of putative Joinvillea ascendens and Kobresia littledalei PPDs with Arabidopsis PPD1.
A. & B. The PPD domain from Arabidopsis aligned with putative PPD domains from J. ascendens (A) and K. littledalei (B). The J. ascendens alignment gave 75% identities and 88% positives, while K. littledalei gave 79% identities and 90% positives. C. The distance between domains is altered in Kobresia and Joinvillea compared to Arabidopsis, a notable feature being the greatly increased spacing between PPD and TIFY domains for the Joinvillea protein in contrast with the Arabidopsis sequence.
Fig 8.
Alignment of PPD, TIFY and Jas* domains of Arabidopsis thaliana (Ath), Amborella trichopoda (Atr), Selaginella moellendorffii (Smo), Sphagnum fallax (Sfa), Sphagnum magellanicum (Sma) and Marchantia polymorpha (Mpo).
A. Over the PPD domain, M. polymorpha is the most divergent sequence from Arabidopsis, with residues conserved in all sequences shown in yellow and residues conserved in all sequences except M. polymorpha shown in blue. B. The aligned TIFY domains. C. The aligned Jas* domains.